Defining Macrophage Polarization Signatures via Bioactive Recombinant Proteins and LEGENDplex™ profiling 2257843

Abstract Introduction Macrophage polarization into pro-inflammatory M1 and anti-inflammatory M2 phenotypes, including M2a (tissue repair), M2b (immune regulation), and M2c (immune suppression and remodeling), is a critical process in immune regulation and tissue homeostasis. Understanding and controlling this polarization is essential for immunological research and therapeutic applications. This study evaluated the effectiveness of bioactive recombinant growth factors (M-CSF and GM-CSF) and cytokines (IL-4, IL-13, and IFN-γ) in directing M1 and M2 macrophage differentiation and polarization from human peripheral blood mononuclear cells. Methods M1 polarization was induced using human recombinant GM-CSF, IFN-γ, and E. coli LPS, while M2 polarization was achieved with human recombinant M-CSF, IL-4, and IL-13. Phenotypic characterization was performed via flow cytometry (FC) and immunocytochemistry (ICC) using markers specific to M1 (iNOS, CD74, CD80) and M2 (CD163, TGM2). Functional validation was conducted using LEGENDplex™ multiplex cytokine profiling with the Human Macrophages/Microglia Panel (IL-12p70, TNFα, IL-6, IL-1β, IL-12p40, IL-23, IFNγ, IP-10, IL-4, IL-10, Arginase, TARC, and IL-1RA) and the Human Tumor-Associated Macrophage (TAM) Panel (MMP-9, CXCL-9, CCL20, CCL18, CD163, MMP-2, Galectin-3, VEGF, M-CSF, PDGF-BB, G-CSF, CCL22, and YKL-40). Results Using directly conjugated antibodies in FC and ICC, the study confirmed successful differentiation of M1 and M2 macrophages through the expression of specific markers. LEGENDplex™ analysis revealed distinct cytokine signatures associated with inflammatory, anti-inflammatory, and regulatory responses, validating the functional polarization of the macrophages. Conclusion These findings demonstrate the reliability of the in-house bioactive recombinant proteins for macrophage differentiation and polarization. They also highlight the utility of specific signature markers, direct-conjugated antibodies, and LEGENDplex™ panels for downstream immunological applications. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)